Name: combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins
Uploaded: 2017-01-06T10:30:00
Description: Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

This work was supported by grant NIH R01 GM097204 (APV and JDH). Funding for JHM was supplied by an HHMI Undergraduate Research Summer Fellowship.

NOTE: A diagram of the conceptual framework or workflow is described in Figure 1 for reference. The protocol can be broken down into four stages: computational or sequence based predictions, protein expression and purification, biochemical characterization, and crystallization. The examples shown analyze Shroom SD2 domains and/or Shroom-Rock complexes, but can be utilized with any protein.
1. Use Established Web-based Tools to Generate Computational Predictions of Coiled-coil Do...

<ol>
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L., Hildebrand, J. D., Harland, R. M., Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom+induces+apical+constriction+and+is+required+for+hingepoint+formation+during+neural+tube+closure.">Shroom induces apical constriction and is required for hingepoint formation during neural tube closure.</a> Curr Biol. 13 (24), 2125-2137 (2003).</li><li>Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom+regulates+epithelial+cell+shape+via+the+apical+positioning+of+an+actomyosin+network.">Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network.</a> J Cell Sci. 118 (Pt 22), 5191-5203 (2005).</li><li>Dietz, M. L., Bernaciak, T. M., Vendetti, F., Kielec, J. M., Hildebrand, J. 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The protocol described here is designed to help the user identify domain boundaries within large coiled-coil proteins to facilitate their crystallization. The protocol relies on a holistic incorporation of a variety of data from computational predictions and other sources to generate a series of potential domain boundaries. These are followed by a set of biochemical experiments which are quick and inexpensive, and are used to further refine these initial hypotheses. Using this approach, the user could quickly eliminate p...

Structure determination via X-ray crystallography has made fundamental contributions to every field of modern biology; providing an atomic view of the macromolecules that support life and how they interact with one another in a variety of contexts; allowing us to understand the mechanisms that cause disease and providing opportunities to rationally design drugs to treat disease. Crystallography has long been the dominant experimental technique for determining macromolecular structure, and currently accounts for 89.3% of the structural database (www.rcsb.org). This technique has many advantages, including the potential for very high resolution, the ability to visualize macromolecules with a broad range of sizes, relatively easy data collection, and the opportunity to visualize how the macromolecule interacts with solvent as well as ligands.
Despite numerous technological improvements in recombinant protein expression1,2, purification3, and molecular biology used to generate these systems4, the single biggest obstacle in the crystallographic process remains the ability to grow diffraction quality crystals. This has been especially true for proteins which contain large coiled-coil domains. It has been estimated that as much as 5% of all amino acids are found within coiled-coils5,6, making this a very common structural feature7, yet these proteins are often more difficult to purify and crystallize than globular proteins8-10. This is further compounded by the fact that coiled-coil domains are often found within the context of a larger protein, therefore correctly predicting the boundaries of these domains is critical to avoid the inclusion of unstructured or flexible sequence that is often detrimental for crystallization.
Here we present a conceptual framework combining web-based computational analyses with experimental data from the bench, to help guide users through the initial stages of the crystallographic process including: how to select protein fragment(s) for structural studies, and how to prepare and characterize protein samples prior to crystallization attempts. We focus our analysis on two proteins containing large coiled-coil domains, Shroom (Shrm) and Rho-kinase (Rock). These proteins were chosen as they both contain coiled-coil domains and are known to form a biologically relevant complex11-16. Shroom and Rho-kinase (Rock) are predicted to contain ~200 and 680 residues of coiled-coil respectively, many portions of which have been characterized structurally17-20. The method described here provides a streamlined workflow to quickly identify fragments of coiled-coil containing protein that will be amenable for crystallization, however, the techniques described can easily be adapted for most protein or protein-complexes or modified to incorporate high-throughput approaches as available. Lastly, these methods are generally inexpensive and can be performed by users at nearly all experience levels.

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			<td height="21" style="height:21px;width:336px;">BL21(DE3) Rosetta</td>
			<td style="width:263px;">Emd Millipore</td>
			<td style="width:136px;">70954-3</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Star</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td style="width:136px;">C601003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Codon Plus</td>
			<td style="width:263px;">Agilent Technologies</td>
			<td style="width:136px;">230245</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">Lysozyme</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">L3008-5GM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ni-NTA resin</td>
			<td style="width:263px;">Life Technologies</td>
			<td style="width:136px;">25216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SubtilisinA</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">S1211-10ML</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">24 well Cryschem Plate</td>
			<td>Hampton research</td>
			<td style="width:136px;">HR3-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INTELLI-PLATE &#160;96:</td>
			<td style="width:263px;">Art Robbins Instruments</td>
			<td style="width:136px;">102-0001-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 3350</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-591</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">PEG 8000</td>
			<td style="width:263px;">Hampton research</td>
			<td style="width:136px;">HR2-515</td>
			<td></td>
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			<td height="21" style="height:21px;width:336px;">PEG 400</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-603</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 4000</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-605</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:336px;">pcDNA3.1-Clover-mRuby2</td>
			<td style="width:263px;">Addgene</td>
			<td style="width:136px;">49089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Overnight Express Autoinduction System 1</td>
			<td style="width:263px;">Emd Millipore</td>
			<td>71300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysogeny Broth powder</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td>12795027&#160;</td>
			<td></td>
		</tr>
	</tbody>
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combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

A diagram depicting the workflow utilized in this system is shown in Figure 1 and includes three main stages. Computational analysis of the sequence is utilized to develop hypotheses about the domain boundaries of the coiled-coil protein of interest. An example of an annotated analysis of the Shrm2 SD2 domain is shown in Figure 2. In this diagram, the goal was to identify possible domain boundaries for a conserved domain at the C-terminus of the cytoskele...

Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

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A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots (Video) | JoVE

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...